KAI1/CD82 is a member of the tetraspanin superfamily and has been re-discovered as a cancer metastasis suppressor. But the mechanism of the KAI1/CD82-mediated suppression of cancer metastasis still remains unclear. Similar to other tetraspanins, KAI1/CD82 has been reported to regulate cell migration and cancer cell invasiveness. Our studies indicate that: 1) restoration of the KAI1/CD82 expression in prostate cancer cells inhibits cell motility, 2) the FAK-p130CAS/CrK-paxillin signaling pathway is required for KAI1/CD82-mediated suppression of prostate cancer cell motility, 3) KAI1/CD82 disrupts the formation of motility-related subcellular structures such as focal adhesion and cortical actin network, 4) KASP (or EWI2/PGRL), a novel Ig superfamily protein, was identified as a KAI1/CD82-binding protein, and 5) KASP (or EWI2/PGRL) functionally coordinates with KAI 1/CD82 in cell migration. Therefore, we hypothesize that the KAI1/CD82-mediated suppression of cancer metastasis depends on its inhibition of cell motility through 1) regulating the intracellular signaling pathways that control cell movement, and 2) participating the transmembrane complex that is involved in cell movement. So, we propose to first define the structural and functional elements in KAI1/CD82 molecule responsible for the inhibition of cell motility and invasiveness. In particular, the roles of specific biochemical features such as acylation, internalization, and KASP (or EWl2/PGRL) association in cancer cell motility and invasiveness will be determined, in order to conclusively link a specific biochemical feature of KAI1/CD82 to its inhibition of cell motility. Second, we will assess the role of KASP (or EWI2/PGRL) in KAI1/CD82-mediated suppression of cancer cell motility and invasiveness. Third, we will assess how the biochemical feature(s) required for the motility-inhibition affects cell motility by analyzing cell motility-related cellular functions such as adhesion, spreading, and vesicle trafficking and cell motility-related molecular signaling. Together, these studies will identify the mechanism responsible for the KAI1/CD82-mediated suppression of cancer metastasis. Understanding the mechanistic roles of KAI1/CD82 in cancer cell motility will promises to lead to the development of therapeutics capable of specially inhibiting cancer metastasis.